LABORATORY EXERCISE 3

ROUTINE PARAFFIN METHOD

Introduction:
A better and more effective means of studying tissues whether normal or abnormal is by
examination of their sections and smears which have been permanently preserved, stained for
demonstration of specific structures, and mounted on glass slides with coverslips for
permanent keeping. Routine paraffin method is the most common method used in the
laboratory.
The first and most critical step in tissue processing is fixation. Its primary aim is to
preserve the morphologic and chemical integrity of the cell in as life-like a manner as possible
and to harden and protect the tissue from the trauma of further handling so that it is easier to
cut during gross examination. In routine paraffin methods, formalin is the most commonly used
fixative.
Tissues contain large amounts of water, both intracellular and extracellular. This water
must be removed so that it may be replaced by wax. The process to accomplish the said step is
known as dehydration. Dehydration is done by using dehydration agents such as alcohol.
The next step is clearing or dealcoholization. It is a process whereby alcohol is removed
from the tissue and replaced by a fluid (clearing agent) that will dissolve the wax with which the
tissue must be impregnated.
Infiltration or impregnation is the process whereby the clearing agent is completely
removed from the tissue and replaced by a medium that will fill all natural cavities, spaces, and
interstices of the tissues, even the spaces within the constituent cells, and that will set to a
sufficiently firm consistency to allow the cutting of suitably thin sections without undue
distortion and without alteration of the spatial relationships of the tissue and cellular elements.
The impregnating medium commonly used is paraffin.
The impregnated tissue is then arranged precisely in a mold containing a medium
(paraffin) and allowed to solidify in a process known as casting or blocking.
Objectives:
After this session, you are expected to:
 practice the proper way of handling specimens for histopathologic examination.
 correctly use the different apparatus and equipment used in tissue processing.
 properly perform the different steps in tissue processing.

Materials:  Paraffin pitcher

 Coplin jars
 fresh tissue  Water bath
 small wide-mouth bottles  Block trimmer/single-edge blade
 scalpel  Paraffin oven
 forceps  Pan of Coldwater
 cutting board  10% buffered neutral formalin
 pencil  70% ethyl alcohol
 tissue cassette/capsule  80% ethyl alcohol
 paper boats/molds  90% ethyl alcohol
 Bunsen burner/alcohol lamp  Absolute ethyl alcohol
  Xylene
 Melted paraffin wax

INFILTRATION
Procedures: 6. Infiltrate tissue in four changes of
liquid paraffin at 15 intervals and
FIXATION allow to remain for 3 hours in the
1. Cut tissue into sections of not more final change of wax. The liquid
than 3mm in thickness. Label and paraffin must be placed in a paraffin
place the tissue in a tissue oven, which is 2ºC to 5ºC higher
capsule/cassette. Use forceps in than the melting point of paraffin.
handling the specimen a. Wax 1 for 15 minutes
2. Place the tissue capsule in a bottle b. Wax 2 for 15 minutes
with 10% buffered neutral formalin, c. Wax 3 for 15 minutes
for 6 – 12 hours. Formalin must be d. Wax 4 for 15 minutes
20 times the volume of the e. Wax 5 for 3 hours
specimen.
3. In another bottle with the same EMBEDDING
amount of 10% buffered formalin, 7. Fill the mold with melted paraffin.
place the tissue for another 6 – 12 8. Warm a pair of blunt-nose forceps
hours. and use them to transfer the tissue
from the paraffin bath to the mold
9. Warm the forceps again and orient
DEHYDRATION the tissue until it is lying in the
4. Subject the fixed tissue in five desired plane.
changes of ascending 10. Remove the corresponding label
grades/concentrations of ethyl from the cassette and place it
alcohol. The amount of alcohol must against the side of the mold
be 10 times the volume of the adjacent to the tissue. The number
tissue. should be placed in such a way that
a. 70% ethyl alcohol for 6 hours is always facing outside.
b. 90% ethyl alcohol overnight 11. Blow on the surface until a thin film
c. Absolute ethyl alcohol 1 for 2 of wax has solidified.
hours 12. Transfer the mold into a container of
d. Absolute ethyl alcohol 2 for 1 cold water and immerse it gently.
hour The mould should remain
e. Absolute ethyl alcohol 3 for 1 submerged until the paraffin
hour hardens which will take 10-30
minutes.
CLEARING
5. Subject the dehydrated tissue to 2 BLOCKING AND TRIMMING
changes of xylene for 1 hour each. 13. Remove the excess wax from the
a. Xylene 1 for 1 hour block so that it forms a 4-sided prism
b. Xylene 2 for 1 hour or until it assumes the shape of the
 block holder and almost exposes the tissue on top.

LABORATORY EXERCISE 3
ROUTINE PARAFFIN METHOD
WORKSHEET

Name: 00

Observations and Discussion:

Figure 1 Figure 2

Fixed tissue Unfixed tissue

In the realm of histopathologic examination, meticulous handling and processing of

specimens are fundamental for accurate diagnostics, necessitating delicate removal to
prevent damage, moisture retention to avert drying, and avoidance of heat and chemical
alterations, alongside the proper use of equipment such as microtomes, cryostats, and tissue
processors (Bruce-Gregorios & Faldas, 2017). The tissue processing steps involve careful
fixation, dehydration, clearing, infiltration, embedding, sectioning, and staining of tissue
samples.

In the laboratory, observing the fixed and unfixed chicken heart liver specimens
reveals a stark contrast in their physical properties. The fixed tissue presents a rubbery
texture and a uniform, somewhat pale appearance, a distinct chemical odor, which are all
indications of the protein cross-linking and pigment homogenization caused by the fixative
(Thavarajah et al., 2012). Conversely, the unfixed specimen is noticeably softer and more
vibrant in color, reflecting its more natural state. Still, it also carries the risk of rapid
degradation, which could compromise its structure and emit an organic, potentially pungent
 odor as decomposition sets in—all of these observations underlie the importance of fixation
in maintaining tissue integrity during examination.

Guide Questions:

A. FIXATION

1. Why is it important to fix the tissue?

In histology, tissue fixation is an essential procedure that keeps cells and tissue
constituents in a "life-like state" for microscopic inspection. It maintains antigenicity,
improves the refractive index of tissue constituents, and inhibits autolysis, necrosis, and
bacterial decomposition. It is necessary to produce uniform physical and chemical
properties in tissue sections so that comparisons, morphological and chemical changes,
and patterns can be observed (Rolls, 2023). Thus, tissue fixation is essential to histology
because it protects the morphology and composition of the tissue, stops degenerative
processes, and creates thin, stained sections that are ready for analysis.

2. What will happen to the tissue if it is not properly fixed?

For specimens to retain their morphological and histological information, proper

tissue fixation is essential (Unity Health and Toronto, 2023). If the tissues are not
properly fixed or there is delayed fixation, bacterial decomposition, tissue digestion, and
autolysis can set in. It is best to use cross-linking, coagulation, or precipitation to stop
the loss and diffusion of soluble materials. In an aqueous environment, fixatives first
cause shrinkage, swelling, and hardening and thus, should be used in proper amounts.
Tissues experience additional alterations in non-aqueous environments, though,
including under-fixation, which results in excessively soft tissue, and over-fixation, which
causes brittleness and difficulty in sectioning. As such, one should aim for appropriate
tissue processing, microtomy, and staining, and avoid over-fixation to prevent these
problems.

3. What is the difference between a fixed and unfixed tissue? In terms of appearance.
based on what you saw during the activity.)

Various differences between fixed and unfixed tissues of their appearance

underscore the critical role of fixation in preserving tissue integrity for examination
purposes. Fixed tissues display a rubbery texture and a consistent, somewhat pale
coloration, along with a distinct chemical scent. Thavarajah et al. (2012) explained that
these attributes stem from the cross-linking of proteins and the homogenization of
pigments induced by the fixation process. Conversely, unfixed specimens are notably
softer and exhibit a more vibrant color, reflecting their closer resemblance to their
natural state. However, they are also friable, meaning they are prone to crumbling or
 breaking apart easily, and may appear hemorrhagic, indicating the presence of bleeding
or blood-like discoloration, particularly in vascular tissues (Titford & Horenstein, 2005).

References:

Bruce-Gregorios, J., & Faldas, M. (2017). Histopathologic techniques. Independently Published.

Rolls, G. (2023). Process of fixation and the nature of fixatives. Intro to Tissue Fixation in
Histology: Types, Methods & More. https://www.leicabiosystems.com/knowledge-
pathway/fixation-and-fixatives-1-the-process-of-fixation-and-the-nature-of-fixatives/?
fbclid=IwAR1Q7sWqVnVRD3MFP-9PE_PlRofOl6F6jPd9XuznWPz1kKLQSPl1BLiZzAg

Thavarajah, R., Mudimbaimannar, V. K., Elizabeth, J., Rao, U. K., & Ranganathan, K. (2012).
Chemical and physical basics of routine formaldehyde fixation. Journal of oral and
maxillofacial pathology, 16(3), 400-405.

Titford, M. E., & Horenstein, M. G. (2005). Histomorphologic assessment of formalin substitute

fixatives for diagnostic surgical pathology. Archives of pathology & laboratory
medicine, 129(4), 502-506.

Unity Health and Toronto. (2023). Tissue fixation and fixatives.

https://research.unityhealth.to/staff-services/research-facilities/facilities/histology-
core/tissue-fixation-and-fixatives/?
fbclid=IwAR2w3KCzQQyfaOMPTwWeEVhaL03JjwaeVuoTmuki890EMBhNYgPds85HLFw
B. DEHYDRATION AND CLEARING

1. What is the purpose of clearing?

In histopathology, clearing is a critical step that involves extracting alcohol from

tissues and substituting it with a fluid that is miscible with wax (Bathari, 2023). Since
dehydrating agents and paraffin wax are incompatible, the clearing agent is crucial.
Tissues treated with clearing agents have a higher refractive index, which increases
transparency. Xylene is the most widely used clearing agent because it is inexpensive
and acts quickly. Other choices include chloroform, which is perfect for nervous tissue,
lymph nodes, and embryos, and toluene, a colorless, flammable liquid with an aromatic
smell. The best clearing agent for large blocks, chloroform evaporatively evaporates
from wax quickly and has minimal shrinkage. Although it is costly and quickly
evaporates, chloroform is also the best clearing agent for lymph nodes, nervous tissue,
and embryos.

2. What will happen to the clearing agent if the tissue immersed is incompletely
dehydrated?

A milky solution is produced when tissue is not completely dehydrated, which

stops the clearing agent from completely replacing the alcohol (Bathari, 2023). This is
because the agent is miscible with paraffin wax and the dehydrating agent, which
prevents the tissue from being transparent and makes it opaque. Thus, before
submerging the tissue in the clearing agent, it is imperative to make sure it is completely
dehydrated.

3. What is the best clearing agent? or dehydrating agent for the routine method?

The type of tissue being processed and the intended use downstream determine
which clearing and dehydrating agents are best. Bruce-Gregorios and Faldas (2017)
recommended ethanol, also known as ethyl alcohol, to be the best dehydrating agent
for routine tissue dehydration. It is a colorless, transparent, and flammable fluid but it is
regarded as the best dehydrating agent because of its quick-acting nature, ability to
combine with water and other organic solvents, and ease of tissue penetration. It is not
harmful, nor is it especially costly.

Xylene is also deemed to be the best clearing agent for routine methods due to
its inexpensiveness and quick action (Bathari, 2023). Bruce-Gregorios and Faldas (2017)
also specified that it is the fastest clearing agent that can clear tissues in 15 to 30
minutes, making it appropriate for urgent biopsies. It is miscible with paraffin and
 absolute alcohol, and it turns tissues transparent. It may be used for celloidin sections
and does not remove aniline dyes. In a paraffin oven, it evaporates fast, and wax is a
simple substitute throughout the impregnation and embedding processes.

References:

Bathari, S. (2023, April 6). Clearing of tissues: Histological techniques. Paramedics World.
https://paramedicsworld.com/histological-techniques/clearing-of-tissues/medical-
paramedical-studynotes?fbclid=IwAR08Z26OqC9jof7veOhw2Y6autIaM-
mvSW2OnNxuA6TF2PeMPbezXxXuoP4

Bruce-Gregorios, J., & Faldas, M. (2017). Histopathologic techniques. Independently Published.

C. INFILTRATION, EMBEDDING, BLOCKING AND TRIMMING

1. What is the purpose of infiltration?

In histopathology, infiltration is a procedure that substitutes a medium that fills

all cavities and tissue spaces in place of clearing agents, making cutting easier (Wilson,
2023). To do this, the tissue is submerged in baths that gradually increase in paraffin
wax concentration. Paraffin wax is infused into the tissue by the infiltration agent,
causing it to solidify and form a wax block that makes thin histological sections easy to
cut. The objective is to eliminate the clearing agents and produce a solid consistency
that simplifies cutting. All things considered, infiltration is an essential step in
histopathology that guarantees precise and effective tissue analysis.

2. What is blocking? Trimming?

In histopathology, a process called "blocking" involves embedding a tissue

sample in a solid material, such as paraffin wax, to produce thin sections that can be
examined under a microscope (Rolls, 2023). A hardened medium is injected into the
dehydrated tissue, causing it to solidify into a block. After slicing the block to reveal the
tissue, a microtome is used to cut thin sections. On the other hand, trimming is an
essential step in histopathology that entails removing extra tissue from a biopsy
specimen so that the tissue is the right size and orientation (Guachi-Guachi et al., 2023).
Tissue samples must be appropriately trimmed after fixation to get the right size and
orientation of the tissue. To get a sample size that is appropriate for later histology
processes like embedding and sectioning, this step is equally crucial.

3. What will happen to the tissue if it is improperly impregnated?

In histopathology, impregnation entails swapping out the clearing agent for a

medium that fills in all of the tissue gaps and cavities. An incomplete filling of tissue gaps
caused by improper impregnation can produce tissue that is either too soft or too brittle
(Bathari, 2018). Overly soft tissue may not stain well and may be difficult to cut into thin
sections. When sectioning, too brittle tissue may break or crack, resulting in the loss of
important data. To prevent these problems, inappropriate impregnation is essential in
histopathology.
References:

Bathari, S. (2018, March 1). Impregnation and tissue embedding: Histopathology notes.
Paramedics World. https://paramedicsworld.com/histological-techniques/impregnation-
tissue-embedding/medical-paramedical-studynotes?
fbclid=IwAR2d3ChfpZTVesUE9Sqrhl5ONpUg2YWLUhxemY6869rpQB612_SM2eUQP6s

Guachi-Guachi, L., Ruspi, J., Scarlino, P., Poliziani, A., Ciancia, S., Lunni, D., ... & Ricotti, L. (2023).
Convolutional neural networks applied to microtomy: Identifying the trimming-end
cutting routine on paraffin-embedded tissue blocks. Engineering Applications of Artificial
Intelligence, 126, 106963.

Rolls, G. (2023). An introduction to specimen processing. Tissue Processing Overview: Steps &
Techniques for Histopathology.
https://www.leicabiosystems.com/knowledge-pathway/an-introduction-to-specimen-
processing/?fbclid=IwAR2v6QCSaa0z2Si01O7brFK-9_eBXsiccvemYtPQ2Cpd-
GWJpMtHNAZuXYg

Wilson, M. (2023, March 15). Tissue processing for histology in 6 easy steps. Bitesize Bio.
https://bitesizebio.com/13469/tissue-processing-for-histology-what-exactly-happens/?
fbclid=IwAR2v6QCSaa0z2Si01O7brFK-9_eBXsiccvemYtPQ2Cpd-GWJpMtHNAZuXYg

Conclusion:

In the laboratory, we have learned about the routine paraffin method in histopathologic
techniques. The objectives were sufficiently achieved as we practiced the proper handling of
specimens for histopathologic examination, correctly utilized various apparatus and
equipment in tissue processing, and properly performed all the steps required in tissue
processing. The laboratory activity has further elucidated the significance of each step:
fixation preserves tissue structure, improper fixation leads to decay, rubbery pale fixed tissue
withstands processes more than the soft bloody unfixed tissue, clearing increases tissue
transparency, incomplete dehydration impedes clearing, ethanol, and xylene are preferred for
routine dehydration and clearing, and finally, infiltration, embedding, blocking, and trimming
that removes excess tissue are essential for preparing the tissue for microscopic examination
—with proper impregnation in all of the tissue’s gaps ensuring the tissue's not too brittle or
soft towards suitability for sectioning. Thus, the routine paraffin method that encompasses
careful fixation, dehydration, clearing, infiltration, embedding, sectioning, and staining of
tissue samples is vital to be mastered—as any improper performance in any of these steps
might waste precious specimens that will also dictate the precious fate of the patient.